Influenza A and B virus infections induce robust innate and adaptive immune responses in the healthy human population, a response that is recapitulated with various animal models, including mice. Although these responses do not usually result in sterilizing, broadly reactive (heterosubtypic) immunity that protects from infection with all influenza strains, they do strongly reduce mortality rates and disease severity following heterosubtypic infections. A non-redundant component of this robust response is the production of ?natural IgM?, spontaneously-produced antibodies that bind broadly to all test influenza virus strains. Natural IgM is produced in the steady-state by classical B-1 cells and by terminally-differentiated B-1- derived plasma cells in spleen and bone marrow and following influenza infection also in the draining lymph nodes by CD5+ B-1 cells mobilized from the pleural cavity. In their absence, or in the absence of the Fc- receptor for IgM (FcR) on B cells, fully protective humoral immunity is not established, mice show increased lung viral loads, and they succumb to infection at increased rates. Thus, natural IgM and the cells responsible for their production are part of the ?broadly protective? immunity to influenza, a complex and diverse response that significantly reduces disease burden and death among those infected. Elucidating what constitutes the most effective, mortality and morbidity-reducing, broad immunity to influenza is of high significance for the design of new therapeutic and prophylactic treatments, and is the long-term objective of this proposal. It is therefore responsive to PA-18-859, which aims to support research dedicated to the development of a universal influenza virus vaccine. Specific Aim 1 Is to determine the signals required for the terminal differentiation of B-1 cells to strongly IgM-secreting plasma cells (B-1PC). Using in vivo cell depletion and cell reconstitution experiments we will identify the signals required for B-1PC development. Gene expression and functional studies will determine the repertoires and anti-influenza activity of natural IgM from B-1PC and B-1 cells, respectively. Specific Aim 2 will investigate the effects of TLR and BCR-mediated signals on B-1 cell responses to influenza. We will test our hypothesis that a two-step process activates CD5+ B-1 cells: TLR-mediated signals induce cell proliferation and loss of the BCR signaling inhibitor CD5, enabling subsequent BCR-signals to drive specific B-1 cell responses. Finally, in Specific Aim 3 we will explore the mechanism underlying the non- redundant function of secreted IgM and of the FcR in enhancing protective T-dependent IgG responses to influenza in vitro and in vivo. Expected results would provide conceptional advances for understanding natural IgM, CD5+ and CD5- B-1 cell function, and the fundamental processes underlying T-dependent B cell activation. The data would identify natural IgM as a critical prophylactic target for enhanced broad humoral immunity to influenza.